Lactococcus intestinalis sp. nov., a new lactic acid bacterium isolated from intestinal contents in Alzheimer’s disease mice

A gram-positive, facultatively anaerobic, and coccoid or ovoid-shaped bacterium designated M2458T was isolated from the intestinal contents of APPswe/PSΔE9 mouse model of Alzheimer’s disease. With the polyphasic approach, the taxonomic position of the novel isolate was confirmed. Strain M2458T grew well at 37 °C on YCFA agar. Strain M2458T belongs to the family Streptococcaceae and class Bacilli, and it is closed to Lactococcus formosensis NBRC 109475T (97.59% sequence similarity) according to its 16S rRNA gene sequence. In a comparison of two housekeeping genes, rpoA and rpoB, strain M2458T was found to be well separated from Lactococcus formosensis NBRC 109475T. On the basis of whole genome sequences, the DNA G+C content was 38.29 mol%. The phylogentic analysis of the whole genome showed that a different branch was clearly formed in the phylogenetic tree of strain M2458T compared to other strains in the genus Lactococcus. A total of eight genes in strain M2458T are involved in the ‘neurodegenerative disease’ pathway, which involves an annotated protein (glyceraldehyde 3-phosphate dehydrogenase) involved in Alzheimer's disease. In terms of average nucleotide identity and digital DNA–DNA hybridization, strain M2458T was identified as a novel species of the genus Lacococcus. The major fatty acids (> 10% of the total fatty acids) were C18:1ω9c (39.68%), C16:0 (13.26%) and C18:1ω7c (11.52%). The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and an unidentified phospholipid. As a result of its genotypic and phenotypic characteristics, strain M2458T was considered to be a new species within the genus Lactococcus; the name Lactococcus intestinalis sp. nov. has been proposed, with type strain M2458T (=JCM 35706 = CGMCC 1.60066).

formosensis NBRC 109475 T (97.59% sequence similarity) according to its 16S rRNA gene sequence. In a comparison of two housekeeping genes, rpoA and rpoB, strain M2458 T was found to be well separated from Lactococcus formosensis NBRC 109475 T . On the basis of whole genome sequences, the DNA G+C content was 38.29 mol%. The phylogentic analysis of the whole genome showed that a different branch was clearly formed in the phylogenetic tree of strain M2458 T compared to other strains in the genus Lactococcus. A total of eight genes in strain M2458 T are involved in the 'neurodegenerative disease' pathway, which involves an annotated protein (glyceraldehyde 3-phosphate dehydrogenase) involved in Alzheimer's disease. In terms of average nucleotide identity and digital DNA-DNA hybridization, strain M2458 T was identified as a novel species of the genus Lacococcus. The major fatty acids (> 10% of the total fatty acids) were C18:1ω9c (39.68%), C16:0 (13.26%) and C18:1ω7c (11.52%). The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and an unidentified phospholipid. As a result of its genotypic and phenotypic characteristics, strain M2458 T was considered to be a new species within the genus Lactococcus; the name Lactococcus intestinalis sp. nov. has been proposed, with type strain M2458 T (=JCM 35706 = CGMCC 1.60066).

Introduction
It is known that lactic acid bacteria (LAB) are widespread in nature and are usually found in foods such as meat, milk and vegetables and their products (Siezen et al. 2002;Dewan Tamang 2007). Furthermore, lactic acid bacteria are highly prevalent in the oral and intestinal tracts of livestock and poultry, as well as in a few clinical cases, where they are important members of the normal microbiota constituting a specific region in the oral and intestinal environments of mammals. The genus Lactococcus is a member of the lactic acid bacteria was proposed by Schleifer et al. as a separate genus distinct from the genus Streptococcus (Schleifer et al. 1985). The genus Lactococcus currently contains twenty-two species and six subspecies with valid names (https:// lpsn. dsmz. de/ genus/ lacto coccus) (Noda et al. 2020). Some members of the genus Lactococcus has been widely used in the industrial manufacturing of fermented dairy products in many countries and regions (Aucouturier et al. 2018). However, Lactococcus garvieae causes hemorrhagic septicemia in fish (Castro et al. 2019), Lactococcus lactis, L. garvieae can also cause infectious diseases such as liver abscess and osteomyelitis in humans (Heo et al. 2019;Antolin et al. 2004;James et al. 2000).
Alzheimer' disease (AD) is the most common form of neurodegenerative disease, which accounts for 60-70% of all dementia cases (Revi 2020;Komura 2022). In the nineteenth century, Metchnikoff hypothesized that lactobacilli play an important role in human health based on observations of the longevity of Bulgarians who consumed large quantities of yogurt (Komura 2022). It is expected that LAB may have protective effects on neurological functions via the brain-gut correlation. In the early stage, we isolated 18 strains of Lactococcus spp. from intestinal contents of APP swe /PS ΔE9 (PAP) mouse model of Alzheimer's disease, and Lactococcus is the dominant genus of PAP mice. Moreover, Stain M2458 was selected as the type strain for the further research. We found some genes of M2458 T were involved in the metabolic pathway of Alzheimer's disease, which demonstrate potential applications in AD prevention and treatment. In this study, we described the polyphasic characterization of strain M2458 T to represent a novel species of the genus Lactococcus.

Strain isolation
On September 17, 2021, strain M2458 T was isolated from the intestinal contents of APP swe /PS ΔE9 mouse model of Alzheimer's disease purchased from Beijing Huafu Kang Biotechnology Co., Ltd. in China. Intestinal contents were collected and immediately put into anaerobic PBS solution containing 1% cysteine, and then filtered through 70 μM filter. The filtrate was plated on YCFA agar by a serial dilution procedure and cultivated for 2 days at 37 °C in anaerobic incubator. The colonies formed were picked out and purified via the subculturing using the same culture conditions. Among them, the ivory strain was detected by 16S rRNA gene PCR, and it was found that the strain was a novel species, with 97.59% similarity to the previously known species. Strain M2458 T which was selected and cultured on YCFA agar at 37 °C and stored in YCFA broth containing 20% glycerol (w/v) at -80 °C.

Phenotypic and biochemical characteristic
The Gram staining procedure was followed as described by Buck (Buck 1982). Cell morphology and the presence of spores were examined by scanning electron microscopy. Using previously described methods (Chen et al. 2014;Varsha Nampoothiri 2016), phenotypic characteristics such as lactic acid fermentation type, salt tolerance and growth temperature range were assessed. According to the manufacturer's instructions, API 50 CHL fermentation kit (bioMe´rieux) was used for acid production from carbohydrates. In addition, the enzymic activities of strain M2458 T was assayed using the API ZYM system (bioMe´rieux). The tests were performed according to the manufacturer's instructions. It was terminated by adding one drop of each of API A and API B ZYM reagents after incubating at 37 °C for 4 h.

Chemotaxonomic analysis
As described by Minnikin et al. (1984), the polar lipids of stain M2458 T were determined. Strain M2458 T and its closest reference strain were also characterized for their cellular fatty acid profiles. The strain was cultured in YCFA agar at 37 °C for 72 h, and the fatty acid was extracted by protocol of the Sherlock Microbial Identification System (MIDI). Then, fatty acid methyl esters were detected by gas chromatography (type 6890; HP) (model 6890; Hewlett Packard) and MIDI system (Sasser 1990).

Genome sequencing and analysis
The genome sequence of strain M2458 T was obtained by using PacBio RSII apparatus at BGI (Beijing Genomics institution). De novo assembly was achieved by using SOAPdenovo v1.05 software. The average nucleotide identity (ANI) values were calculated by the OrthoANI using EzBioCloud (www. ezbio cloud. net/ tools/ ani), using the algorithm described by Yoon et al. (2017). The digital DNA-DNA hybridization (dDDH) values were calculated using Genome-to-Genome Distance Calculator (GGDC) version 3.0 (http:// ggdc. dsmz. de/ ggdc. php) (Auch et al. 2010;Meier-Kolthoff et al. 2013). With the use of FastTree, the phylogenomic tree was constructed using a concatenated alignment of 120 conserved bacterial single-copy genes with GTDB-Tk v. 1.5.1 (Parks et al. 2018;Yang et al. 2022).

Physiological characteristics
Colonies of strain M2458 T were cream-coloured, opaque, round and convex on YCFA plates. Scanning electron microscopy analysis revealed coccoid or ovoid-shaped cells present singly or in short chains (Fig. 1). No spores and mycelium were observed and cells could not grow in a medium with 4% (w/v) NaCl. The tolerance to 4% NaCl clearly differentiated strain M2458 T from Lactococcus formosensis NBRC 109475 T . Besides, M2458 T grew well at pH 5.0 but Lactococcus formosensis NBRC 109475 T did not. Acids of M2458 T were not produced from Mannitol and Amygdalin, while the acid of Lactococcus Fig. 1 Scanning electron microscope of strain M2458 T formosensis NBRC 109475 T and Lactococcus garviae subsp. garviae NBRC 100934 T were produced from them M2458 T could be differentiated from the reference strains by a combination of temperature and pH for growth, acid production from various substrates, and enzymatic activities. Detailed phenotypic and differential properties are presented in Table 1.

Phylogenetic tree analysis
The 16S rRNA gene sequence of strain M2458 T has been deposited in the GenBank database with the accession number of OM926013. The EzBiocloud server showed that the 16S rRNA sequence of strain M2458 T has less than 97.59% homology with only one related taxon of the genus Lactococcus. The novel isolate showed the highest sequence similarities to Lactococcus formosensis NBRC 109475 T (97.59%) and Lactococcus garviae subsp. garviae NBRC 100934 T (97.10%). The phylogenetic tree analysis indicated that strain M2458 T had a separate branch within the genus Lactococcus and formed a group with Lactococcus formosensis NBRC 109475 T (Fig. 2). Therefore, Lactococcus formosensis NBRC 109475 T was selected as the closest reference strain for comparative analysis with strain M2458 T .
Housekeeper genes are now commonly used to supplement 16S rRNA gene analysis to determine species levels. Comparing rpoA gene sequence with GenBank database sequence, strain M2458 T (OP672167) has the highest similarity (96.56%) to Lactococcus formosensis NBRC 109475 T (AB780470) and the second highest similarity (96.53%) to Lactococcus garviae subsp. garviae NBRC 100934 T (JN226492). Figure S1 shows the phylogenetic analysis of the rpoA gene. In addition, the rpoB gene was compared with that of other type strains of species of the genus Lactococcus. The results showed that strain M2458 T (OP672168) has 93.57% sequence similarity to Lactococcus formosensis NBRC 109475 T (GU799685). The phylogenetic analysis of rpoB gene is shown in Figure S2.

Chemotaxonomic analysis
The cellular fatty acids of strain M2458 T and its closest reference strains are shown in Table S1. The major fatty acids of strain M2458 T were C18:1ω9c, C16:0 and C18:1ω7c. Strain M2458 T and its phylogenetically closest relatives exhibited some qualitative and quantitative differences in fatty acid content. PE, PG, and DPG were the major polar lipids, while 1 unidentified polar lipid was the minor polar lipid (Fig. 3). The major polar lipids in the type strains Lactococcus formosensis NBRC 109475 T and Lactococcus garviae subsp. garviae NBRC 100934 T were DPG and GL, while PE was the minor polar lipid. Fig. 2 Maximum Likelihood tree of M2458 T and other related latococci based on 16S rRNA sequences. Bacillus subtilis NCIB 3610 T was used as an outgroup. *Branches that were also found using the maximum-parsimony method (Fitch 1971); + Branches that were also found using the Neighbourjoining (Felsenstein 1985); * + Branches that were found using all three methods. Numbers at nodes are percentage bootstrap values based on 1000 replicates, only values above 50% are given. Bootstrap values are indicated at branch points based on 1000 iterations. Bar, 0.01 substitutions per nucleotide position. GenBank accession numbers are given in parentheses Genome analysis The genome sequence was submitted to the GenBank (www. ncbi. nlm. nih. gov/) database (JAKZEZ000000000). The genome size of strain M2458 T was 4,171,867 bp and 3866 genes were annotated. Among them, 79 genes were tRNAs and 5 genes were rRNAs. Total DNA G+C content of strain M2458 T was determined to be 38.29 mol%, which was within the G+C content range of the genus Lactococcus (35.5-47.5 mol%) (Chen et al. 2014(Chen et al. , 2013. In the phylogenomic tree (Fig. 4), strain M2458 T clustered with the members of the genus Lactococcus but clearly formed different branch. The ANI values of strain M2458 T with the closely species Lactococcus formosensis NBRC 109475 T , Lactococcus garviae subsp. garviae NBRC 100934 T and Lactococcus petauri 159469 T were 82.35, 82.45 and 82.46%, respectively, and the dDDH values estimated by the GGDC were 26.9, 26.8 and 26.4%, respectively (Table 2). In general, species boundaries for ANI and dDDH values are 95-96% and 70%, respectively (Chun et al. 2018).
For strain M2458 T , total 2485 genes (40.3%) and 2136 genes (34.6%) were functionally annotated within COG database and KEGG database, respectively (Fig. 5A). The predicted genes distributed among 23 COG categories dominated by translation, ribosomal structure and biogenesis (13.9%), carbohydrate transport and metabolism (10.7%), general function prediction only (8.9%), transcription (8.0%), and amino acid transport and metabolism (7.6%) ( Fig. 5B). In addition, 111 genes are involved in lipid metabolism (4.5%). However, studies have shown that Alzheimer's disease is related to lipid metabolism (Sun et al. 2022). The predicted gene annotation by KEGG database which resulted in total 30 functional categories (Fig. 5C). 8 genes of strain M2458 T are involved in 'neurodegenerative disease' pathway, among them the annotated protein involved in Alzheimer's disease was glyceraldehyde 3-phosphate dehydrogenase. Glyceraldehyde 3-phosphate dehydrogenase also exists in Lactococcus garviae subsp. garviae NBRC 100934 T . It is well known that Alzheimer's disease is a senile neurological disease. 7 genes of strain M2458 T are involved in 'aging' pathway. Age related superoxide dismutase also exists in Lactococcus garviae subsp. garviae NBRC 100934 T .

Taxonomic conclusion
In accordance with the data, strain M2458 T displays numerous features that are common to members of the genus Lactococcus, e.g., PG and DPG as major lipids; and C18:1ω9c, C16:0 and C18:1ω7c as major fatty acids. However, chemotaxonomic and phenotypic differences differentiate the novel isolate from previously recognized species of the genus Lactococcus despite the phylogenetic tree of the 16S rRNA gene sequence is in the same group (Table 1). Therefore, strain M2458 T should be assigned to the genus Lactococcus with the proposed name Lactococcus intestinalis sp. nov. Lactococcus intestinalis (intestinal' is. L.gen. n. intestinalis of intestinal, where the type strain M2458 T was isolated).
Cells are Gram-stain-positive, catalase-negative, coccoid or ovoid-shaped, facultatively anaerobic and grow well anaerobically on YCFA agar at 37 °C. Cells utilize d-glucose homofermentatively but do not produce gas. L-lactic acid is the main product. Growth occurs at 15-45 °C. The cells can grow at pH 5-7 and have a tolerance for NaCl from 0 to 3%. The enzyme reaction is positive for leucine aminopeptidase, acid phosphatase, but is negative for β-Glucuronidase. Acid is produced from ribose, arbutin, d-glucose, d-fructose, d-mannose, salicin, cellobiose, maltose, trehalose, starch, gentiobiose, sucrose, d-tagatose, N-acetylglucosamine, aesculin, but is not produced from amygdalin, l-arabinose, d-galactose, d-mannitol, lactose,  Author contributions Conceived and designed the experiments: S.L. and Q.C.. Performed the experiments: S.P., L.X., and S.W.. Analyzed the data: S.P., L.X., L.M. and Z.L.. Worte and revised the paper: S.P. and S.L. All authors reviewed the manuscript.

Availability of data and material
The GenBank accession numbers for 16S rRNA gene sequences of strains M2458 T is OM926013. The draft genome sequences of strains M2458 T has been deposited at NCBI under the accession no. JAKZEZ000000000.

Conflict of interest
The authors declare that there is no conflict of interest.

Ethics approval
The animal study protocol was approved by the Ethics Committee of the Laboratory of Animal Science of Peking Union Medical College (protocol code SL21001).